This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the reporting period, we showed that co-delivery of GM-CSF DNA with the DNA prime for a SHIV-89.6 DNA/MVA vaccine enhanced control of high dose intrarectal SHIV-89.6P challenges. Here we tested whether GM-CSF serves as an adjuvant for protection against a repeat low dose heterologous SIV challenge. Groups of 8 rhesus macaques (all A*01negative, one B*08 and one B*17 positive per group) were inoculated intramuscularly at 0 and 8 weeks with 3mg of SIV239 DNA, which did or did not co-express rhesus GM-CSF, and boosted with SIV239 MVA at 16 and 24 weeks. Both the DNA and MVA immunogens expressed Gag, PR, RT and Env. Twelve weekly low dose intrarectal challenges with 5000 TCID50 (1.8x107 copies of viral RNA) of SIV E660 were initiated 20 weeks following the final MVA inoculation. Protection against infection was plotted using the Kaplan-Meier method showing a nonparametric cumulative plot. The GM-CSF-adjuvanted immunizations increased protection against acquisition from 25% in the non adjuvanted group (6/8 infected) to 70% in the adjuvanted group (2/7 infected). Protection in the adjuvanted group was significantly different from protection of the controls (9/9 infected)(p=0.003). Co-expressed GM-CSF did not affect the titers of anti-Env binding Ab or the magnitudes or polyfunctionality of elicited CD4 and CD8 T cells. It did enhance the titers of neutralizing Ab for a neutralization sensitive variant of E660 (E660.11) (p=0.02), the avidity of anti-Env Ab for the 239 Env (p=0.02) and showed a trend for increased avidity for the E660 Env (0.09) and the frequency of anti-Env IgA in rectal secretions. The GM-CSF-adjuvanted protection did not extend to reductions in peak levels of viral RNA. Both infected macaques had excellent viral pull down. Results demonstrated that GM-CSF expressed in cis can provide strong adjuvant activity for prevention of acquisition of a heterologous challenge by a SIV DNA/MVA vaccine.